Joint Sites Research Articles

Abstract PR05: Genome-wide screening identifies novel YAP modulators

Abstract The transcriptional coactivator YAP is the key downstream effector of the Hippo pathway, which is a central regulator of organ size, stem cell/progenitor cell expansion, and tissue regeneration. Diverse studies have shown the importance of a precise regulation of this pathway and its key downstream regulator YAP for tissue homeostasis. To achieve new insights in the Hippo-independent transcriptional regulation of YAP in breast cancer, we set out to identify new YAP modulators using a genome-wide CRISPR screen and Perturb-Seq. We identified the transcriptional repressor protein Trichorhinophalangeal Syndrome 1 (TRPS1) as a potent repressor of YAP-dependent transactivation, using a genome-wide CRISPR screen. TRPS1 globally regulates YAP-dependent transcription by binding to a large set of joint genomic sites, mainly enhancers. Moreover, TRPS1 antagonizes activating YAP/TEAD complexes by recruiting corepressor complexes to joint sites. Loss of TRPS1 leads to activation of enhancers due to increased H3K27 acetylation and an altered promoter-enhancer interaction landscape. TRPS1 is commonly amplified in breast cancer, suggesting that restrained YAP activity favors tumor growth. Combined, we uncover TRPS1 as an epigenetic regulator of YAP activity in breast cancer. Additionally, we developed a Trps1-deficient mouse line that we will use to test the in vivo role of Trps1 in mammary gland physiology and breast cancer. Furthermore, we combined CRISPR-based gene perturbation with droplet-based single-cell RNA sequencing (Perturb-Seq), to perform a second large-scale analysis on potential YAP modulators. To this end, we used a CRISPR-library containing 158 previously from us in a Bio-ID assay identified YAP interactors. Together, we uncover several novel YAP modulators, contributing to a better understanding of the Hippo-independent transcriptional regulation of YAP. This abstract is also being presented as Poster B40. Citation Format: Dana Elster, Marie Tollot, Karin Schlegelmilch, Alessandro Ori, Andreas Rosenwald, Erik Sahai, Björn von Eyss. Genome-wide screening identifies novel YAP modulators [abstract]. In: Proceedings of the AACR Special Conference on the Hippo Pathway: Signaling, Cancer, and Beyond; 2019 May 8-11; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(8_Suppl):Abstract nr PR05.

The contribution of age and obesity to the number of painful joint sites in individuals reporting osteoarthritis: a population-based study.

ObjectiveTo investigate the association of OA risk factors with number of painful joint sites in a representative population sample.MethodsAnalysis of the 2009 Survey on Living with Chronic Diseases in Canada – Arthritis Component (n = 1614) for respondents reporting symptomatic OA. Variables: painful joints sites (hands, wrists, elbows, shoulders, hips, knees, ankles, feet, back, neck), joint symptom duration, sociodemographic characteristics, smoking, comorbidities and BMI. Zero-truncated negative binomial regressions were used to investigate the association between number of painful joint sites and the variables. Generalizability of findings was assessed by a similar analysis in a clinical hip/knee OA sample.ResultsThe sample comprised 73% women and 56% were aged <65 years. The mean number of painful joint sites was 3.8: 84% reported pain at ≥2 sites, and 45% at ≥4 sites. Age, BMI, education and smoking were not associated with the number of joint sites. Significant associations were found with being female [rate ratio (RR) = 1.23, 95% CI 1.09, 1.39], having more comorbidities (RR = 1.11, 95% CI 1.07, 1.15) and longer symptom duration (RR = 1.16, 95% CI 1.09, 1.24), although the increase in joint sites with duration was small. Similar regression results were found with the clinical OA sample.ConclusionThe lack of an association of age and BMI (obesity) with number of painful joint sites in OA raises questions about the role of these risk factors and our understanding of OA as a multi-joint disease. Filling this knowledge gap is critical to making progress with defining OA phenotypes and identifying potential aetiological mechanisms.

Blame it on the knee: the association of hand osteoarthritis and obesity

Purpose: Obesity and overweight are recognized risk factors for osteoarthritis (OA). There is a strong and consistently found relationship between high body mass index (BMI) and knee OA, but there is ongoing uncertainty as to whether higher BMI is a risk factor for hand OA. As with most OA research, the studies on hand OA consider this joint in isolation (i.e. without consideration of other joints). Given that hand OA often occurs in conjunction with OA of other joints, a failure to consider other joints could lead to misleading results. The Canadian Longitudinal Study on Aging (CLSA) asked separately about the presence of knee, hip, and hand OA, providing the opportunity to investigate whether there is an independent association between hand OA and BMI, accounting for the association of BMI with other concurrent joints, particularly the knee. Methods: The CLSA began in 2013 and is following a representative sample of respondents aged 45-85 years at baseline to gain insight into the development of disease and disability through the aging process. Respondents in the baseline assessment were asked separately about OA in the knee, in the hip, and in the hand, with the lead-in question, “Has a doctor ever told you that you have⋯” Participant height and weight was measured and BMI (kg/m2) calculated, and sociodemographic information was collected. Participants reporting arthritis other than OA or without information on OA or BMI were excluded from the present study. Descriptive analyses compared BMI distributions between those without OA, and those with OA of the knee, hip and hand, individually and with each possible combination of OA at these joint sites. Visual inspection was used to determine whether the shape of the BMI distributions differed for combinations of hand OA with knee and hip OA. The relationship between BMI and joint site was quantified by linear regression, adjusted for age and sex. The relationship between obesity and joint site was quantified by logistic regression, adjusted for age and sex. Results: The analytic sample included 25,459 individuals. Overall, 24.9% of the sample reported OA at any site. Among these, 56.6% reported knee OA, 31.2% hip OA and 46.9% hand OA. Forty-seven percent of individuals reporting hand OA also reported knee and/or hip OA. There was minimal difference in mean BMI between the OA groups (Table, Column A), but the percent obese, especially for the combinations of individual joint sites, showed greater differences across groups (Table, Column B). The distributions of BMI for the hand OA and hand and hip OA groups were similar, but distinct from the distributions that included knee OA, which were likewise similar but had a higher proportion of individuals in the obese I and II/III categories (Figure). Findings for males and females and different age groups were similar. Regression results for OA overall and for OA at individual joint sites (without consideration of other joints), showed a positive and significant association between each joint group and higher BMI and, in particular, likelihood of being obese (Table, Columns C and D). However, when all combinations of joint sites were simultaneously considered, neither hand OA alone nor hand and hip OA together were significantly associated with higher BMI or obesity, whereas any combinations that included knee OA were significantly associated with higher BMI and, in particular, likelihood of being obese (Table, Column C and D). Conclusions: Hand OA assessed without regard to the involvement of other joints appears to be significantly associated with higher BMI and obesity. However, when considering OA in other joints, and in particular knee OA, an independent association between hand OA and BMI/obesity was not found. This suggests that positive associations seen in previous studies are likely due to the presence, but lack of consideration, of concomitant knee OA. Furthermore, the results suggest that a simple comparison of mean BMI values, even when age and sex adjusted, between OA groups may miss important differences that exist in the upper tails of the BMI distribution (i.e. the obese portion of the distribution) where there is greater risk for OA. The findings also raise questions as to whether obesity is a risk factor for OA in general, or only for knee OA.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Characterization of the pathophysiological processes of osteoarthritis using RNA sequencing data of subchondral bone

Purpose: Osteoarthritis (OA) has a considerable genetic component and genetic research has resulted in many insights into underlying disease pathways. Most recent large-scale genomic association studies have identified TGFA , MGP, CHADL as compelling genes involved in the aetiology of OA. The functions of these genes confirm that deviations in both cartilage and bone maintenance processes are major pathways underlying OA pathology in humans. Moreover, follow-up studies have shown that risk SNPs frequently modulate pathology due to altering transcription of the genes in cis both in bone and cartilage. Nonetheless, follow up study of these genes towards underlying biological mechanisms, preclinical studies on target discovery and eventually drug testing have thus far focused on gene expression profiling in articular cartilage. The aim of the current study was to characterize interactive pathophysiological processes of subchondral bone and articular cartilage in human osteoarthritis (OA) in large human cohort RNA sequencing. Methods: We performed RNA sequencing on macroscopically preserved and lesioned OA subchondral bone of patients that underwent a joint replacement surgery due to OA (N=24 pairs; 6 hips, 18 knees, RAAK-study). Unsupervised hierarchical clustering and differential expression analysis were performed on the data. Gene enrichment was performed using the online tool DAVID. Moreover, we compared the differentially expressed genes identified here to our previously reported differentially expressed genes in OA articular cartilage. Results: Upon performing cluster analysis we identified two clusters; one representing hips and the other representing knees. Due to the low hip sample size, differential expression analysis was performed on knee samples only. As shown in Figure 1, we identified 1757 significantly differentially expressed genes between lesioned and preserved subchondral bone. The most significantly downregulated gene was FRZB (FC=0.48, FDR=3.07x10-12), encoding the frizzled receptor protein and is a well-known cartilage OA gene. The most significantly upregulated gene was CNTNAP2 (FC=2.90, FDR=3.38x10-6), encoding the contactin-associated protein-like 2 protein (CASPR2). Among the 1757 differentially expressed genes, 102 genes showed an absolute foldchange of 2 or higher. The highest upregulated gene was STMN2 (FC=23.0, FDR=1.8x10-4), encoding stathmin 2, while the most downregulated gene was CHRDL2 (FC=0.09, FDR=4.13x10-6), encoding chordin-like protein 2 and is also known to be involved in OA in cartilage. Enrichment of the differentially expressed genes resulted in significant enrichment of GO-terms regarding the extracellular matrix. To investigate whether the subchondral bone and the articular cartilage show similarities in the OA pathophysiological processes, we compared the results presented here with our previously reported results on differentially expressed genes in OA articular cartilage. As a result, we identified 398 genes that were differentially expressed in both tissues, of which 361 showed similar directions of effect, including the genes IL11, encoding Interleukin 11, and CHADL, encoding Chondroadherin-Like Protein. Conclusions: To our knowledge, we are the first to report on the differential expression pattern of OA subchondral bone using RNA sequencing on a large sample size while integrating these with differential expression patterns of cartilage of the same individuals. By performing cluster analysis on our RNA sequencing data, we identified two clusters representing hips and knees, respectively. These clusters indicate distinct OA pathophysiological processes in the subchondral bone between the two joint sites, which was not previously seen with similar analyses of the cartilage. Among the most consistently differentially expressed genes with OA pathophysiology in bone and cartilage are IL11, and CHADL. Notably, these genes were recently found in large GWA studies on OA indicating also their causal involvement in the OA pathophysiology. Moreover, IL11 was recognized as being targeted by FDA approved drugs. As such, we advocate that IL11 could be an attractive potential drug for OA patients with a therapeutic effect both in cartilage and subchondral.

P118 Orthopaedic surgical interventions in a cohort of patients with hypermobility related disorders, compared with chronic pain syndrome patients in a tertiary referral centre

Abstract Background Mechanisms of pain associated with hypermobility are poorly understood and include nociceptive pain due to structural joint changes, neuropathic pain due to disrupted proprioception, muscle weakness and central sensitisation. The influence of anxiety and low mood is also unknown. There is limited published evidence regarding orthopaedic interventions in hypermobile patients particularly following the updated 2017 Ehlers-Danlos syndrome (EDS) classification criteria. We aimed to evaluate the characteristics of a hypermobility patient cohort who had received surgical interventions and compare these with a patient population with chronic pain syndromes. Methods A retrospective analysis of medical records was conducted of patients attending a tertiary referral centre (University College London Hospital) between January 2018 and December 2018. A hypermobility-related disorders cohort was compared with a cohort of patients with chronic pain syndrome, including fibromyalgia (but without any evidence of hypermobility). Results There were 350 patients (300 females, 50 males) in the hypermobility cohort with a mean age of 36 years (range 18-71 years) at time of clinic review. 63% had hypermobility spectrum disorder or hypermobility syndrome and 37% had EDS (hypermobile, classic, vascular and other rare types). 24% of hypermobile patients had undergone orthopaedic interventions. The Beighton score did not correlate with surgical procedures. 134 patients (121 females, 13 males) with chronic pain syndromes were identified, with a mean age of 51 years (range 26-85 years). 16% of chronic pain patients had received orthopaedic surgery. 76% of hypermobile patients who had orthopaedic interventions were under 50 years old, compared to 23% in the chronic pain cohort. The total number of surgical procedures in the hypermobility cohort was 227 (0.6485 interventions per patient) compared with 25 in the chronic pain cohort (0.1865 interventions per patient); relative risk 3.477 (95% CI 2.42-4.99; P &amp;lt; 0.0001, chi-squared test). 33% of hypermobile patients had surgery on two or more joint groups (including 8 patients who had surgery in four or more joint groups), whereas 95% of chronic patients had surgery on only one joint group. In the hypermobile group the knee (23%) and hips (24%) were the most common site of operative intervention; however in the chronic pain cohort the most common sites were the knee (32%), spine (20%) and shoulders (20%), with only 4% requiring hip surgery. Conclusion Patients with hypermobility have a greater number of surgical procedures at multiple joint sites and at a younger age than patients with chronic pain syndromes, suggesting that mechanical pathology (rather than pain alone) and anticipated surgical success may contribute to the need for surgical intervention. The Beighton score does not appear to be a reliable predictor of surgical intervention. Earlier diagnosis and a holistic, non-operative approach, including physiotherapy and pain management, may reduce the need for surgical procedures. Disclosures S. Wright None. P. Mehta None. A. Rahman None. H. Kazkaz None.

Risk Factor Profiles for Individuals With Diagnosed OA and With Symptoms Indicative of OA: Findings From the Canadian Longitudinal Study on Aging.

ObjectiveThe vast majority of published estimates of osteoarthritis (OA) burden are based on an OA diagnosis. These data are limited, as individuals often do not visit a physician until their symptoms are moderate to severe. This study compared individuals with an OA diagnosis to those with OA joint symptoms but without a diagnosis considering a number of sociodemographic and health characteristics. A further distinction was made between individuals with symptoms in one joint site and those with symptoms in multiple joint sites.MethodsData are from 23 186 respondents aged 45 to 85 years from the first cycle of the Canadian Longitudinal Study on Aging. A multinomial logistic regression model examined the relationship between sociodemographic‐ and health‐related characteristics and OA status (diagnosed OA, joint symptoms without OA, no OA or joint symptoms). In addition, logistic regression models assessed the relationship between OA status and usually experiencing pain and having some degree of functional limitation.ResultsTwenty‐one percent of respondents reported a diagnosis of OA, and 25% reported symptoms typical of OA but without an OA diagnosis. Other than being slightly younger, the characteristic profile of individuals with symptoms in two or more joint sites was indistinguishable from that of those with diagnosed OA.ConclusionIt may be warranted to consider OA‐like multiple joint symptoms when deriving estimates of OA‐attributed population health and cost burden.

Sleep Quality Is Related to Worsening Knee Pain in Those with Widespread Pain: The Multicenter Osteoarthritis Study.

We examined the association between sleep and odds of developing knee pain, and whether this relationship varied by status of widespread pain (WSP). At the 60-month visit of the Multicenter Osteoarthritis Study, sleep quality and restless sleep were each assessed by using a single item from 2 validated questionnaires. Each sleep measure was categorized into 3 levels, with poor/most restless sleep as the reference. WSP was defined as pain above and below the waist on both sides of the body and axially using a standard homunculus, based on the American College of Rheumatology criteria. Outcomes from 60-84 months included (1) knee pain worsening (KPW; defined as minimal clinically important difference in WOMAC pain), (2) prevalent, and (3) incident consistent frequent knee pain. We applied generalized estimating equations in multivariable logistic regression models. We studied 2329 participants (4658 knees; 67.9 yrs, body mass index 30.9]. We found that WSP modified the relationship between sleep quality and KPW (p = 0.002 for interaction). Among persons with WSP, OR (95% CI) for KPW was 0.53 (0.35-0.78) for those with very good sleep quality (p trend < 0.001); additionally, we found the strongest association of sleep quality in persons with > 8 painful joint sites (p trend < 0.01), but not in those with ≤ 2 painful joint sites. Similar results were observed using restless sleep, in the presence of WSP. The cross-sectional relationship between sleep and prevalence of consistent frequent knee pain was significant. Better sleep was related to less KPW with coexisting widespread pain.

Time origin and structural analysis of the induced CRISPR/cas9 megabase-sized deletions and duplications involving the Cntn6 gene in mice

In a previous study using one-step CRISPR/Cas9 genome editing in mouse zygotes, we created five founders carrying a 1,137 kb deletion and two founders carrying the same deletion, plus a 2,274 kb duplication involving the Cntn6 gene (encoding contactin-6). Using these mice, the present study had the following aims: (i) to establish stage of origin of these rearrangements; (ii) to determine the fate of the deleted DNA fragments; and (iii) to estimate the scale of unpredicted DNA changes accompanying the rearrangements. The present study demonstrated that all targeted deletions and duplications occurred at the one-cell stage and more often in one pronucleus only. FISH analysis revealed that there were no traces of the deleted DNA fragments either within chromosome 6 or on other chromosomes. These data were consistent with the Southern blot analysis showing that chromosomes with deletion often had close to expected sizes of removed DNA fragments. High-throughput DNA sequencing of two homozygotes for duplication demonstrated that there were no unexpected significant or scale DNA changes either at the gRNA and joint sites or other genome sites. Thus, our data suggested that CRISPR/Cas9 technology could generate megabase-sized deletions and duplications in mouse gametes at a reasonably specific level.

Optimized coagulation pathway of Al13: Effect of in-situ Aggregation of Al13

The coagulation mechanism for removing particles by Al13 has been extensively investigated for water treatments. It was widely accepted that Al13 played important roles in coagulation mainly by charge neutralization and electrostatic patch. However, the discovery of Al13 aggregates (Al13agg) in flocs indicated that the real coagulation process should be different from the previous understanding, including when Al13agg were generated and how it interacted with negative particles. The aggregation process of Al13 during coagulation and its micro-interfacial effect on particle coagulation remains to be explored. In this study, to investigate the aggregation of Al13 and its effect on coagulation performance, two parallel coagulation jar tests were conducted on silica suspensions by preformed Al13agg and Al13, respectively. The results showed that optimized coagulation for particle removal by Al13 occurred from pH 7 to pH 9, which was dominated by the in-situ aggregation of Al13. The results confirmed that Al13agg were both present in flocs generated in two tests, however, the morphology and distribution of surface Al of flocs were different for two tests. The in-situ formed Al13agg covered all over the silica particles in flocs, resulting in compact structure with rough surfaces, while the preformed Al13agg mainly distributed on joint sites between particles, generating denser flocs with smooth surfaces. This difference verified that the in-situ aggregation of Al13 was the key factor to optimized particle coagulation. The overall optimized particle coagulation by Al13 should undergo the following pathway: charge neutralization – in-situ aggregation of Al13 – inter-particle bridging.

Joint hypermobility is not positively associated with prevalent multiple joint osteoarthritis: a cross-sectional study of older adults

BackgroundThis cross-sectional study evaluated associations of joint hypermobility and multiple joint osteoarthritis (MJOA) in a community-based cohort of adults 45+ years of age.MethodsMJOA and joint hypermobility data were from 1677 participants (mean age 69 years, 68% women) who completed research clinic visits during 2003–2010. Prevalent MJOA was defined in four ways. Radiographic OA (rOA) was defined as Kellgren-Lawrence (KL) > 2 at any included study joint; symptomatic OA (sxOA) required both symptoms and rOA in a joint. Joint hypermobility was defined as a Beighton score of > 4. Separate logistic regression models were used to estimate odds ratios (OR) between joint hypermobility and each MJOA definition, adjusting for age, sex, race, body mass index, and baseline visit.ResultsIn this cohort, 4% had Beighton score > 4 and 63% met any definition of MJOA. Joint hypermobility was associated with significantly lower odds of radiographic and symptomatic MJOA-1 (multiple joint OA-definition 1: involvement of > 1 IP (interphalangeal) nodes and > 2 sites of hip, knee, and spine; 74 and 58% lower, respectively). However, for the other MJOA definitions (i.e., MJOA-2:involvement of > 2 IP joints, > 1 carpometacarpal [CMC] joints, and knee or hip sites; MJOA-3: involvement of > 5 joint sites from among distal interphalangeal, proximal interphalangeal, CMC, hip, knee, or spine sites; and MJOA-4:involvement of > 2 lower body sites (hip, knee, or spine), there were no statistically significant associations. For associations between site-specific hypermobility and any MJOA definition, most adjusted ORs were less than one, but few were statistically significant.ConclusionsOverall, joint hypermobility was not positively associated with any definition of prevalent MJOA in this cohort, and an inverse association existed with one definition of MJOA. Longitudinal studies are needed to determine the contribution of hypermobility to the incidence and progression of MJOA outcomes.

Association IL1Bgene polymorphisms (RS1143634 AND RS1143627) with knee osteoarthritis (KOA) risk in the north Indian population

Purpose: Association IL1B gene polymorphisms (rs1143634 and rs1143627) with knee osteoarthritis (KOA) risk in the North Indian population Methods: We studied the association between IL1B polymorphisms (rs1143634 and rs1143627) and KOA risk by analysing the genotypes of 300 knee OA cases and equal number of controls using polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) method. Results: There was no significant difference in demographic characteristics between the cases and controls. The frequency of genotype rs1143634 and rs1143627 polymorphism of IL1b genes of case and control were compared, no significant difference was found. The frequency of T allele was found significantly greater than C allele (p=0.02) in rs1143634 in cases when compared with normal healthy control showing increased risk in of OA Knee. However, the frequency of C allele was not found significantly greater than T allele (p=0.06) in rs1143627 in cases when compared with normal healthy control. Logistic regression analysis after adjusting for gender and age revealed, no significant differences in the genotype and allele frequencies of both polymorphisms of IL1B gene. No significant difference was observed within the genotype with KL grading and Clinical scores (Womac features and Visual Analogue scale). Further Haplotype approach was also used but no significant difference was observed in any haplotype. Conclusions: In this study both polymorphisms (rs1143634 and rs1143627) of IL1B genes were not significantly associated but marginally positive association data observed for rs1143634 polymorphism in knee cases. When haplotype analysis was performed and no significant difference was found, suggesting that the original associations could be false positive for rs1143634 polymorphism. Further studies are necessary to identify the association of this rs1143634 polymorphism with knee OA in other population and also on different joint sites to give a global view of this polymorphism in pathogenesis of OA.

Orthofix mini external fixator combined with lever technique for minimally invasive prying reduction and limited internal fixation in the treatment of metacarpal and phalangeal fractures at joint sites

Objective To investigate the clinical efficacy of Orthofix mini external fixator combined with lever technique for minimally invasive prying reduction and limited internal fixation in the treatment of metacarpal and phalangeal fractures at joint sites. Methods From October 2014 to June 2017, we applied mini external fixator technique to pull and restore joint alignment, then lever technique for minimally invasive prying reduction to restore articular surface, and limited internal fixation to treat 36 metacarpal and phalangeal fractures at joint sites in 34 cases. Fracture healing and hand function recovery were assessed. Results All the patients obtained short-term follow-up for 6 to 10 months, of which 21 cases obtained long-term follow-up for 10 to 30 months. All the fractures healed after operation. The healing time was 5 to 12 weeks, with an average of 7.3 weeks. According to TAM criteria, the excellent and good rate of short-term follow-up was 82.4%, and the excellent and good rate of long-term follow-up was 90.5%. 5 patients had moderate finger activity during short-term follow-up, of which 2 were reoperated after 10 months and had good long-term follow-up function. One patient was unable to cooperate with functional exercise, and the follow-up showed poor finger activity. Conclusion The method is effective in the treatment of metacarpal and phalangeal fractures at joint sites. This technique is minimally invasive, which does not increase the soft tissue adhesion of joint. The reliable fixation can meet the needs of early functional exercise. The injured hand has good appearance without obvious surgical scar at joint sites and the incidence of hand joint stiffness is low. Key words: Metacarpal bones; Finger phalanges; Fractures,bone; Mini external fixator

TRPS1 shapes YAP/TEAD-dependent transcription in breast cancer cells

Yes-associated protein (YAP), the downstream transducer of the Hippo pathway, is a key regulator of organ size, differentiation and tumorigenesis. To uncover Hippo-independent YAP regulators, we performed a genome-wide CRISPR screen that identifies the transcriptional repressor protein Trichorhinophalangeal Syndrome 1 (TRPS1) as a potent repressor of YAP-dependent transactivation. We show that TRPS1 globally regulates YAP-dependent transcription by binding to a large set of joint genomic sites, mainly enhancers. TRPS1 represses YAP-dependent function by recruiting a spectrum of corepressor complexes to joint sites. Loss of TRPS1 leads to activation of enhancers due to increased H3K27 acetylation and an altered promoter–enhancer interaction landscape. TRPS1 is commonly amplified in breast cancer, which suggests that restrained YAP activity favours tumour growth. High TRPS1 activity is associated with decreased YAP activity and leads to decreased frequency of tumour-infiltrating immune cells. Our study uncovers TRPS1 as an epigenetic regulator of YAP activity in breast cancer.

The role of ultrasound-defined tenosynovitis and synovitis in the prediction of rheumatoid arthritis development.

ObjectivesTenosynovitis (TS) is common in early arthritis. However, the value of US-defined TS in predicting RA development is unclear. We assessed the predictive utility of US-defined TS alongside US-defined synovitis and clinical and serological variables in a prospective cohort of early arthritis patients.MethodsOne hundred and seven patients with clinically apparent synovitis of one or more joint and symptom duration ⩽3 months underwent baseline clinical, laboratory and US assessment of 19 bilateral joint sites and 16 bilateral tendon compartments. Diagnostic outcome was determined after 18 months, applying the 2010 ACR/EULAR classification criteria for RA. The predictive values of US-defined TS for persistent RA were compared with those of US-defined synovitis, clinical and serological variables.ResultsA total of 4066 US joint sites and 3424 US tendon compartments were included in the analysis. Forty-six patients developed persistent RA, 17 patients developed non-RA persistent disease and 44 patients had resolving disease at follow-up. US-defined TS in at least one tendon compartment at baseline was common in all groups (RA 85%, non-RA persistent disease 71% and resolving 70%). On multi-variate analysis, US-defined digit flexor TS provided independent predictive data over and above the presence of ACPA and US-defined joint synovitis.ConclusionUS-defined digit flexor TS provided independent predictive data for persistent RA development in patients with early arthritis. The predictive utility of this tendon site should be further assessed in a larger cohort; investigators designing imaging-based predictive algorithms for RA development should include this tendon component as a candidate variable.

Enhancement of density and ionic conductivity for garnet-type Li5La3Ta2O12 solid electrolyte by self-consolidation strategy

Garnet-type Li5La3Ta2O12 (LLTaO) solid electrolyte is a potential candidate component for future all-solid-state batteries due to its extraordinary stability against the reaction with molten lithium. In contrast with traditional cold isostatic pressing (CIP) method, which generally pursues ultra-high pressure, this paper tries to enhance the density and ionic conductivity of LLTaO by self-consolidation strategy without the assistance of any pressing operations. A LLTaO bulk with a relative density of 95% is obtained. SEM images reveal that the bulk sample is assembled by large dense particles in size of tens of microns indicating that the interstitial space among the particles has been dramatically minimized. Accordingly, the total ionic conductivity and the bulk ionic conductivity at 30 °C are promoted up about one order of magnitude higher to 2.63 × 10−5 S cm−1 and 1.41 × 10−4 S cm−1, respectively. Moreover, the lithium ionic migration network in the crystalline unit cell of LLTaO is first explored from its assembled way. A hexagon-like basic unit with tetrahedral Li1 joint sites and Li1- - Li1 edges is identified. The tetrahedral Li1 sites act as crucial junctions for the transportation of lithium ions. This work would significantly stimulate the development of LLTaO electrolyte membrane technology.

Generation of megabase-scale deletions, inversions and duplications involving the Contactin-6 gene in mice by CRISPR/Cas9 technology

BackgroundCopy Number Variation (CNV) of the human CNTN6 gene (encoding the contactin-6 protein), caused by deletions or duplications, is responsible for severe neurodevelopmental impairments, often in combination with facial dysmorphias. Conversely, deleterious point mutations of this gene do not show any clinical phenotypes. The aim of this study is to generate mice carrying large deletions, duplications and inversions involving the Cntn6 gene as a new experimental model to study CNV of the human CNTN6 locus.ResultsTo generate large chromosomal rearrangements on mouse chromosome 6, we applied CRISPR/Cas9 technology in zygotes. Two guide RNAs (gRNAs) (flanking a DNA fragment of 1137 Mb) together with Cas9 mRNA and single-stranded DNA oligonucleotides (ssODN) were microinjected into the cytoplasm of 599 zygotes of F1 (C57BL x CBA) mice, and 256 of them were transplanted into oviducts of CD-1 females. As a result, we observed the birth of 41 viable F0 offspring. Genotyping of these mice was performed by PCR analysis and sequencing of PCR products. Among the 41 F0 offspring, we identified seven mice with deletions, two animals carrying duplications of the gene and four carrying inversions. Interestingly, two F0 offspring had both deletions and duplications. It is important to note that while three of seven deletion carriers showed expected sequences at the new joint sites, in another three, we identified an absence of 1–10 nucleotides at the CRISPR/Cas9 cut sites, and in one animal, 103 bp were missing, presumably due to error-prone non-homologous end joining. In addition, we detected the absence of 5 and 13 nucleotides at these sites in two F0 duplication carriers. Similar sequence changes at CRISPR/Cas9 cut sites were observed at the right and left boundaries of inversions. Thus, megabase-scale deletions, duplications and inversions were identified in 11 F0 offspring among 41 analyzed, i.e., approximately 25% efficiency. All genetically modified F0 offspring were viable and able to transmit these large chromosomal rearrangements to the next generation.ConclusionsUsing CRISPR/Cas9 technology, we created mice carrying megabase-scale deletions, duplications, and inversions involving the full-sized Cntn6 gene. These mice became founders of new mouse lines, which may be more appropriate experimental models of CNV in the human 3p26.3 region than Сntn6 knockout mice.

Novel joint selection methods can reduce sample size for rheumatoid arthritis clinical trials with ultrasound endpoints.

To determine whether novel methods of selecting joints through (i) ultrasonography (individualized-ultrasound [IUS] method), or (ii) ultrasonography and clinical examination (individualized-composite-ultrasound [ICUS] method) translate into smaller rheumatoid arthritis (RA) clinical trial sample sizes when compared to existing methods utilizing predetermined joint sites for ultrasonography. Cohen's effect size (ES) was estimated (ES^) and a 95% CI (ES^L, ES^U) calculated on a mean change in 3-month total inflammatory score for each method. Corresponding 95% CIs [nL(ES^U), nU(ES^L)] were obtained on a post hoc sample size reflecting the uncertainty in ES^. Sample size calculations were based on a one-sample t-test as the patient numbers needed to provide 80% power at α = 0.05 to reject a null hypothesis H0 : ES = 0 versus alternative hypotheses H1 : ES = ES^, ES = ES^L and ES = ES^U. We aimed to provide point and interval estimates on projected sample sizes for future studies reflecting the uncertainty in our study ES^S. Twenty-four treated RA patients were followed up for 3 months. Utilizing the 12-joint approach and existing methods, the post hoc sample size (95% CI) was 22 (10-245). Corresponding sample sizes using ICUS and IUS were 11 (7-40) and 11 (6-38), respectively. Utilizing a seven-joint approach, the corresponding sample sizes using ICUS and IUS methods were nine (6-24) and 11 (6-35), respectively. Our pilot study suggests that sample size for RA clinical trials with ultrasound endpoints may be reduced using the novel methods, providing justification for larger studies to confirm these observations.

Surveillance of Extrapulmonary Nontuberculous Mycobacteria Infections, Oregon, USA, 2007-2012.

Limited data are available describing extrapulmonary nontuberculous mycobacteria (NTM) infections in the general population. We describe results from statewide population-based laboratory surveillance in Oregon, USA, during 2007–2012. We defined a case of extrapulmonary NTM infection as >1 isolate from skin/soft tissue, disseminated sites, lymph node, joint, or other sites. The annual incidence of extrapulmonary NTM infection (other than Mycobacterium gordonae) was stable, averaging 1.5 cases/100,000 population. Median age of the 334 patients was 51 years, and 53% of patients were female. Half of cases were caused by M. avium complex, but rapid-growing NTM species accounted for one third of cases. Most extrapulmonary NTM infections are skin/soft tissue. Compared with pulmonary NTM infection, more extrapulmonary infections are caused by rapid-growing NTM species. the designation of NTM as a reportable disease in Oregon in 2014 will result in better detection of changes in the incidence and patterns of disease in the future.

Reliability Analysis of a Wearable Sensor Patch (WSP) to Monitor ECG Signals

Abstract Flexible Hybrid Electronic (FHE) devices interface flexible sensors and circuits with conventional rigid electronic components. This work reports preliminary results for the reliability aspects of a project aimed at fabricating a Wearable Sensor Patch (WSP) to monitor ECG signals. The device was fabricated by interfacing flexible electroplated Cu circuit lines and an ECG sensor on a Kapton® polyimide (PI) substrate with rigid electronics connected using SnPb solder (reflow temperature: 204 °C), making it a FHE device. Phase I of this project faced reliability issues as Cu circuit lines were susceptible to failure due to cracking near the front-end signal conditioning chip. This issue needed to be resolved in Phase II of the project to produce a robust device fit to be used in real world applications. The effect of changes in Cu trace thickness (2 and 6 μm) and Kapton® PI thickness (2 and 5 mil) on device robustness was tested. Effect of the use of low reflow temperature SnBi solder (reflow temperature: 175 °C) on device reliability was also tested. Multiple devices fabricated using different configurations of Cu trace and Kapton® PI thicknesses and either SnPb or SnBi solder were bend tested to single out the most robust configuration. Improved solder pad design for Cu traces at solder joint sites was also tested. It was observed that only devices with 6 μm thick Cu traces, 2 mil thick Kapton® and SnBi solder had no defects as a result of thermal cycling during fabrication. They also performed best during bend testing. Some of the factors contributing to robustness of this configuration might be lower CTE mismatch due to lower solder reflow temperature as well as greater strength under bending due to increased thickness. Improved solder pad design for Cu traces also improved device robustness considerably.

Cartilage matrix remodelling differs by disease state and joint type.

Dramatic alterations in mechanical properties have been documented for osteoarthritic (OA) cartilage. However, the matrix composition underlying these changes has not been mapped and their aetiology is not entirely understood. We hypothesised that an understanding of the cartilage matrix heterogeneity could provide insights into the origin of these OA-related alterations. We generated serial transverse cryo sections for 7 different cartilage conditions: 2 joint sites (knee and hip), 2 disease states (healthy and OA) and 3 tissue depths (superficial, middle and deep). By laser capture microscopy, we acquired ~200 cartilage matrix specimens from territorial (T) and interterritorial (IT) regions for all 7 conditions. A standardised matrix area was collected for each condition for a total of 0.02 ± 0.001 mm3 (corresponding to 20 µg of tissue) from a total of 4800 specimens. Extracted proteins were analysed for abundance by targeted proteomics. For most proteins, a lower IT/T ratio was observed for the OA disease state and knee joint type. A major cause of the altered IT/T ratios was the decreased protein abundance in IT regions. The collagenase-derived type III collagen neo-epitope, indicative of collagen proteolysis, was significantly more abundant in OA cartilage. In addition, it was enriched on average of 1.45-fold in IT relative to T matrix. These results were consistent with an elevated proteolysis in IT regions of OA cartilage, due to degenerative influences originating from synovial tissue and/or produced locally by chondrocytes. In addition, they offered direct evidence for dynamic remodelling of cartilage and provided a cogent biochemical template for understanding the alterations of matrix mechanical properties.